Heparan sulfate proteoglycans (HSPGs) contain one or more heparan sulfate chains covalently linked to a protein core. HSPGs are localized to the cell surface and in the extracellular matrix. Due to their strong negative charge, the heparan sulfate chains bind to and modulate interactions between various protein ligands that mediate cell proliferation, including growth factors, morphogens, other extracellular matrix proteins, enzymes, and enzyme inhibitors. HSPGs have been implicated in cell growth and proliferation and tend to be mis-expressed in cancer cells and tumors. Previous studies have indicated that structure of the heparan sulfate chains (the number and arrangement of sulfate groups and uronic acid epimers) and the level of expression of HSPGs can affect tumor growth. The overall goal of this proposal is to uncover and characterize novel genes other than those encoding known heparan sulfate biosynthetic enzymes, whose expression influences heparan sulfate-mediated regulatory networks in cancer progression. To accomplish this goal, we plan to: (1) develop a high-throughput, genome-wide, short-hairpin RNA screen that detects genes involved in heparan sulfate biosynthesis, and (2) identify and characterize candidate genes for their capacity to block tumor cell growth and viability. A screening assay will be designed employing toxin-based screens used previously to identify small molecule inhibitors of heparan sulfate. We plan to search for genes, that when their expression is reduced or eliminated, will confer resistance to heparan sulfate-dependent cytotoxins. A genome-wide shRNA library will be used to knock down gene expression in HeLa (cervical carcinoma) cells. Following knockdown, we will assay for cell viability and identify those candidate genes that conferred resistance to the heparan sulfate-dependent toxin. Analysis of heparan sulfate structure and binding properties, as well as expression analysis of the core proteins will follow. Additionally, we will determine the ability of tumor cells expressing the candidate shRNAs to form colonies in soft agar and induce the formation of tumors in animals. The combined results from the work outlined in this proposal will give us a better understanding of the role heparan sulfate structure and function plays in tumorigenesis. The factors we identify could reveal novel targets for anti-cancer therapies, as well as lead us to methods to manipulate heparan sulfate and its activities in other cell types. PUBLIC HEALTH RELEVANCE: The studies outlined here seek to uncover and characterize novel genes whose expression influences heparan sulfate-mediated regulatory networks in tumor growth. A better understanding of heparan sulfate structure and function as well as identification of factors involved in determining its biological activities could reveal novel targets for anti-cancer therapies. In addition, the potential findings might also give us insight into the mechanisms by which heparan sulfate can influence many other cellular processes that go awry in human pathologies.